This invention is generally in the field of peptide-based vaccines to control treat, or reduce the risk of atherogenic activity in the circulatory system of humans and other animals. In particular, this invention provides compositions and methods for providing means to inhibit the activity of endogenous cholesteryl ester transfer protein (CETP) to treat cardiovascular disease prophylactically or therapeutically or to modulate the relative levels of lipoproteins to produce a condition correlated with a reduced risk of cardiovascular disease, such as atherosclerosis.
Cholesterol circulates through the body predominantly as components of lipoprotein particles (lipoproteins), which are composed of a protein portion, called apolipoproteins (Apo) and various lipids, including phospholipids, triglycerides, cholesterol and cholesteryl esters. There are ten major classes of apolipoproteins: Apo A-I, Apo A-II, Apo-IV, Apo B-48, Apo B-100, Apo C-I, Apo C-II, Apo C-III, Apo D, and Apo E. Lipoproteins are classified by density and composition. For example, high density lipoproteins (HDL), one function of which is to mediate transport of cholesterol from peripheral tissues to the liver, have a density usually in the range of approximately 1.063-1.21 g/mL HDl. contain various amounts of Apo A-I, Apo A-II, Apo C-I, Apo C-II, Apo C-III, Apo D, Apo E, as well as various amounts of lipids, such as cholesterol, cholesteryl esters, phospholipids, and triglycerides.
In contrast to HDL, low density lipoproteins (LDL), which generally have a density of approximately 1.019-1.063 g/ml contain Apo B-100 in association with various lipids. In particular, the amounts of the lipids, cholesterol and cholesteryl esters are considerably higher in LDL than in HDL, when measured as a percentage of dry mass LDL are particularly important in delivering cholesterol to peripheral tissues.
Very low density lipoproteins (VLDL) have a density of approximately 0.95-1.006 g/ml and also differ in composition from other classes of lipoproteins both in their protein and lipid content. VLDL generally have a much higher amount of triglycerides than do HDL or LDL and are particularly important in delivering endogenously synthesized triglycerides from liver to adipose and other tissues. The features and functions of various lipoproteins have been reviewed (see, for example, Mathews, C. K. and van Holde, K. E., Biochemistry, pp. 574-576, 626-630 (The Benjamin/Cummings Publishing Co., Redwood City, Calif., 1990); Havel, R. J., et al., et al., xe2x80x9cIntroduction: Structure and metabolism of plasma lipoproteinsxe2x80x9d, In The Metabolic Basis of Inherited Disease, 6th ed., pp. 1129-1138 (Scriver, C. R., et al., eds.) (McGraw-Hill, Inc., New York, 1989); Zannis, V. I., et al., xe2x80x9cGenetic mutations affecting human lipoproteins, their receptors, and their enzymesxe2x80x9d, In Advances in Human Genetics, Vol. 21, pp. 145-319 (Plenum Press, New York, 1993)).
Decreased susceptibility to cardiovascular disease, such as atherosclerosis, is generally correlated with increased absolute levels of circulating HDL and also increased levels of HDL relative to circulating levels of lower density lipoproteins such as VLDL and LDL (see, e.g., Gordon, D. J., et al., N. Engl. J. Med., 321: 1311-1316 (1989); Castelli, W. P., et al., J. Am. Med. Assoc., 256: 2835-2838 (1986); Miller, N. E., et al., Am. Heart J., 113: 589-597 (1987); Tall, A. R., J. Clin. Invest., 89: 379-384 (1990); Tall, A. R., J. Internal Med., 237: 5-12 (1995)).
Cholesteryl ester transport protein (CETP) mediates the transfer of cholesteryl esters from HDL to TG-rich lipoproteins such as VLDL and LDL, and also the reciprocal exchange of TG from VLDL to HDL (Tall, A. R., J. Internal Med., 237: 5-12 (1995); Tall, A. R., J. Lipid Res., 34: 1255-1274 (1993); Hesler, C. B., et al., J. Biol. Chem., 262: 2275-2282 (1987); Quig, D. W. et al., Ann. Rev. Nutr., 10: 169-193 (1990)). CETP may play a role in modulating the levels of cholesteryl esters and triglyceride associated with various classes of lipoproteins. A high CETP cholesteryl ester transfer activity has been correlated with increased levels of LDL-associated cholesterol and VLDL-associated cholesterol, which in turn are correlated with increased risk of cardiovascular disease (see, e.g., Tato, F., et al., Arterioscler. Thromb. Vascular Biol., 15: 112-120 (1995)).
Hereinafter, LDL-C will be used to refer to total cholesterol including cholesteryl esters and/or unesterified cholesterol associated with low density lipoprotein. VLDL-C will be used to refer to total cholesterol including cholesteryl esters and/or unesterified cholesterol associated with very low density lipoprotein. HDL-C will be used to refer to total cholesterol including cholesteryl esters and/or unesterified cholesterol associated with high density lipoprotein.
CETP isolated from human plasma is a hydrophobic glycoprotein having 476 amino acids and a molecular weight of approximately 66,000 to 74,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gels (Albers. J. J., et al., Arteriosclerosis, 4: 49-58 (1984); Hesler, C. B., et al., J. Biol. Chem., 262: 2275-2282 (1987); Jarnagin, S. S., et al., Proc. Natl. Acad. Sci. USA, 84: 1854-1857 (1987)). A cDNA encoding human CETP has been cloned and sequenced (Drayna, D., et al., Nature, 327: 632-634 (1987)). Polymorphism in human CETP has recently been reported and may be associated with disease in lipid metabolism (Fumeron et al., J. Clin. Invest., 96: 1664-1671 (1995); Juvonen et al., J. Lipid Res., 36: 804-812 (1995)). CETP has been shown to bind CE, TG, phospholipids (Barter, P. J. et al., J. Lipid Res., 21:238-249 (1980)), and lipoproteins (see, e.g., Swenson, T. L., et al., J. Biol. Chem., 264: 14318-14326 (1989)). More recently, the region of CETP defined by the carboxyl terminal 26 amino acids, and in particular amino acids 470 to 475, has been shown to be especially important for neutral lipid binding involved in neutral lipid transfer (Hesler, C. B., et al., J. Biol. Chem., 263: 5020-5023 (1988)), but not phospholipid binding (see, Wang, S., et al., J. Biol. Chem., 267: 17487-17490 (1992); Wang, S., et al., J. Biol. Chem., 270: 612-618 (1995)).
A monoclonal antibody (Mab), TP2 (formerly designated 5C7 in the literature), has been produced which inhibits completely the cholesteryl ester and triglyceride transfer activity of CETP, and to a lesser extent the phospholipid transfer activity (Hesler, C. B., et al., J. Biol. Chem., 263: 5020-5023 (1988)). The epitope of TP2 was localized to the carboxyl terminal 26 amino acids, i.e., the amino acids from arginine-451 to serine-476, of the 74,000 dalton human CETP molecule (see, Hesler, C. B., et al., (1988)). TP2 was reported to inhibit both human and rabbit CETP activity in vitro and rabbit CETP in vivo (Yen, F. T., et al., J. Clin. Invest., 83: 2018-2024 (1989) (TP2 reacting with human CETP); Whitlock et al., J. Clin. Invest., 84: 129-137 (1989) (TP2 reacting with rabbit CETP)). Further analysis of the region of CETP bound by TP2 revealed that amino acids between phenylalanine-463 and leucine-475 are necessary for TP2 binding and for neutral lipid (e.g., cholesteryl ester) transfer activity (see, Wang, S., et al., 1992).
A number of in vivo studies utilizing animal models or humans have indicated that CETP activity can affect the level of circulating cholesterol-containing HDL. Increased CETP cholesteryl ester transfer activity can produce a decrease in HDL-C levels relative to LDL-C and/or VLDL-C levels which in turn is correlated with an increased susceptibility to atherosclerosis. Injection of partially purified human CETP into rats (which normally lack CETP activity), resulted in a shift of cholesteryl ester from HDL to VLDL, consistent with CETP-promoted transfer of cholesteryl ester from HDL to VLDL (Ha, Y. C., et al., Biochim. Biophys. Acta, 833: 203-211 (1985); Ha, Y. C., et al., Comp. Biochem. Physiol., 83B: 463-466 (1986); Gavish, D., et al., J. Lipid Res., 28: 257-267 (1987)). Transgenic mice expressing human CETP were reported to exhibit a significant decrease in the level of cholesterol associated with HDL (see, e.g., Hayek, T., et al., J. Clin. Invest., 90: 505-510 (1992); Breslow, J. L., et al., Proc. Natl. Acad. Sci. USA, 90: 8314-8318 (1993)). Furthermore, whereas wild-type mice are normally highly resistant to atherosclerosis (Breslow, J. L., et al., Proc. Natl. Acad. Sci. USA, 90: 8314-8318 (1993)), transgenic mice expressing a simian CETP were reported to have an altered distribution of cholesterol associated with lipoproteins, namely, elevated levels of LDL-C and VLDL-C and decreased levels of HDL-C (Marotti K. R., et al., Nature, 364: 73-75 (1993)). Transgenic mice expressing simian CETP also were more susceptible to dietary-induced severe atherosclerosis compared to non-expressing control mice and developed atherosclerotic lesions in their aortas significantly larger in area than those found in the control animals and having a large, focal appearance more typical of those found in atherosclerotic lesions in humans (Marotti et al., id). Intravenous infusion of anti-human CETP monoclonal antibodies (Mab) into hamsters and rabbits inhibited CETP activity in vivo and resulted in significantly increased levels of HDL-C levels, decreased levels of HDL-triglyceride, and increased HDL size; again implicating a critical role for CETP in the distribution of cholesterol in circulating lipoproteins (Gaynor, B. J., et al., Atherosclerosis, 110: 101-109 (1994) (hamsters); Whitlock, M. E., et al., J. Clin. Invest., 84: 129-137 (1989) (rabbits)).
CETP deficiency has also been studied in humans. For example, in certain familial studies in Japan, siblings that were homozygous for non-functional alleles of the CETP gene had no detectable CETP activity. Virtually no atherosclerotic plaques were exhibited by these individuals, who also showed a trend toward longevity in their families (see, e.g., Brown, M. L., et al., Nature, 342: 448-451 (1989); Inazu, A., et al., N. Engl. J. Med., 323: 1234-1238 (1990); Bisgaier, C. L., et al., J. Lipid Res., 32: 21-23 (1991)). Such homozygous CETP-deficient individuals also were shown to have an anti-atherogenic lipoprotein profile as evidenced by elevated levels of circulating HDL rich in cholesteryl ester, as well as overall elevated levels of HDL, and exceptionally large HDL, i.e., up to four to six times the size of normal HDL (Brown, M. L., et al., 1989, p. 451). The frequency of this mutation in Japan is relatively high, and may account for an elevated level of HDL in a significant fraction of the Japanese population.
The above studies indicate that CETP plays a major role in transferring cholesteryl ester from HDL to VLDL and LDL, and thereby in altering the relative profile of circulating lipoproteins to one which is associated with an increased risk of cardiovascular disease (e.g., decreased levels of HDL-C and increased levels of VLDL-C and LDL-C). Marotti et al. (above) interpreted their data as indicating that a CETP-induced alteration in cholesterol distribution was the principal reason that arterial lesions developed more rapidly in transgenic, CETP-expressing mice than in non-transgenic control mice when both groups were fed an atherogenic diet. Taken together, the current evidence suggests that increased levels of CETP activity may be predictive of increased risk of cardiovascular disease. Modulation or inhibition of endogenous CETP activity is thus an attractive therapeutic method for modulating the relative levels of lipoproteins to reduce or prevent the progression of or to induce regression of cardiovascular diseases, such as atherosclerosis.
It would be advantageous, therefore, to discover compounds and methods to control CETP activity which would be helpful in preventing or treating cardiovascular disease. To be an effective pharmacological therapeutic, a compound when administered to a significant majority of recipients, ideally, would not elicit an immune response which neutralizes the beneficial activity or effect of the therapeutic compound, must not promote a hypersensitive state in the individual receiving the therapeutic compound, and must not produce untoward side effects. It would also be advantageous if such compounds and methods avoided the necessity for continuous or frequently repeated treatments.
This invention provides compounds and methods useful for the modulation or inhibition of cholesteryl ester transfer protein (CETP) activity. In particular, vaccine peptides are described which, when administered to a mammal raise an antibody response against the mammal""s own endogenous CETP thereby promoting a prophylactic or therapeutic effect against cardiovascular disease, such as atherosclerosis. Such vaccine peptides comprise a helper T cell epitope portion comprising a xe2x80x9cuniversalxe2x80x9d or xe2x80x9cbroad rangexe2x80x9d immunogenic helper T cell epitope, linked, preferably covalently, to a B cell epitope portion comprising one or more B cell epitopes from CETP, such as found in the carboxyl terminal portion of human CETP protein that is involved in a neutral lipid binding or a transfer activity of CETP. Other B cell epitopes from CETP may also be used. Preferably, the B cell epitopes from CETP used in the B cell epitope portion of the vaccine peptides of this invention induce antibodies to endogenous CETP (autoreactive antibodies) which either block CETP function or lead to clearance of circulating CETP in the blood. In addition, the B cell epitopes used in the vaccine peptides of this invention preferably do not also comprise a T cell epitope of CETP so that the possibility of T cell-mediated autoimmune liver damage is avoided.
The vaccine peptides of this invention include various xe2x80x9cmultivalentxe2x80x9d embodiments. For example, multivalent peptides present the immune system with more than a single universal or broad range helper T cell or B cell epitope. Such multivalent vaccine peptides include those which have multiple (two or more) copies of the same or different universal or broad range immunogenic helper T cell epitope and/or multiple copies of the same or different B cell epitope from the CETP protein. Those peptides having more than one unique B cell epitope to which different antibodies can bind may promote the formation of immune complexes to effectively clear CETP from the circulatory system.
In a preferred embodiment, the helper T cell epitope portion of a vaccine peptide of this invention is derived from an amino acid sequence of a universally (broad range) immunogenic helper T cell epitope, such as those found in tetanus and diptheria toxoids, or in antigenic peptides known from pertussis vaccine, Bacile Calmette-Guerin (BCG), polio vaccine, measles vaccine, mumps vaccine, rubella vaccine, purified protein derivative (PPD) of tuberculin, Mycobacterium tuberculosis hsp-70, and keyhole limpet hemocyanin. Furthermore, various universal (or broad range) antigenic helper T cell epitopes may be linked to one another to form multiple (i.e., multivalent) universal antigenic helper T cell epitope portions of the vaccine peptides of this invention.
In a more preferred embodiment of a vaccine peptide of this invention, an amino terminal cysteine residue is covalently linked to an amino acid sequence of a broad range or universal antigenic helper T cell epitope of tetanus toxoid forming the sequence C Q Y I K A N S K F I G I T E (amino acids 1 to 15 of SEQ ID NO:2), which is covalently linked to a B cell epitope portion of a vaccine peptide having the carboxyl terminal CETP amino acid sequence F G F P E H L L V D F L Q S L S (amino acids 16 to 31 of SEQ ID NO:2).
In another preferred embodiment, a multivalent vaccine peptide comprises an amino acid sequence of a broad range or universal antigenic helper T cell epitope of tetanus toxoid, which in turn is covalently linked to a B cell epitope portion consisting of two B cell epitopes of CETP. In one such preferred embodiment of this invention, the multivalent vaccine peptide has the amino acid sequence of SEQ ID NO:8: C Q Y I K A N S K F I G I T E L F P R P D Q Q H S V A Y T F E E D I F G F P E H L L V D F L Q S L S in which an amino terminal cysteine is linked to a T cell epitope from tetanus toxoid (amino acids 2 to 15 of SEQ ID NO:8) linked to an amino acid sequence containing two B cell epitopes of human CETP, i.e., amino acids 349 to 367 and amino acids 461 to 476 of the amino acid sequence for mature human CETP (SEQ ID NO:4). In still another preferred embodiment of this invention, a multivalent vaccine peptide of this invention contains B cell epitopes from the homologous regions of the rabbit CETP (i.e., amino acids 350 to 368 and 481 to 496 of SEQ ID NO:6) and has the amino acid sequence of SEQ ID NO:9: M Q Y I K A N S K F I G I T E R F P R P D G R E A V A Y R F E E D I F G F P K H L L V D F L Q S L S, in which an amino terminal methionine is linked to a T cell epitope from tetanus toxoid (amino acids 2 to 15 of SEQ ID NO:8) which is linked to an amino acid sequence containing the two B cell epitopes from rabbit CETP.
The peptides of this invention may also be linked to one another via a bifunctional linker molecule or a peptide linker molecule having minimal or no immunogenicity. In addition, the peptides may be linked to a common molecule to form peptide assemblies in which multiple copies of the peptides are arranged close to one another. Such multicopy (multivalent) peptide assemblies may be more immunogenic, that is, produce a more effective immune response to endogenous CETP than vaccines comprising unassociated individual peptides.
The vaccine compounds of this invention also may be used in combination with a pharmaceutically acceptable adjuvant.
The immunogenic vaccine peptides of this invention elicit the production of antibodies that are reactive with or recognize endogenous CETP. Administration of vaccine peptides to test animals resulted in a decline in the relative levels of total cholesterol and HDL-C and resulted in a decrease in the development of atherosclerotic lesions. The elicited endogenous anti-CETP antibodies thus promote a physiological condition correlated with decreased risk of cardiovascular disease, and they appear to have a direct effect on preventing or decreasing the formation of atherosclerosis plaques.